Overview

  • Product nameAnti-beta Actin antibody
    See all beta Actin primary antibodies
  • Description
    Rabbit polyclonal to beta Actin
  • Tested applicationsIHC-Fr, IP, WB, ICC, Flow Cyt, IHC-FrFl, IHC-P, IHC-P, ICC/IF, ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Chicken, Guinea pig, Cow, Dog, Human, Pig, Xenopus laevis, Fruit fly (Drosophila melanogaster), Fish, Monkey, Zebrafish, Rhesus monkey, Chinese Hamster
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human beta Actin aa 1-100. The exact sequence is proprietary.
    (Peptide available as ab28691, ab13772)

  • Positive control
    • WB: Mouse brain tissue lysate and HeLa, A431, HEK293, NIH3T3 and PC12 whole cell lysates. IHC-P: Normal human colon and normal human liver tissues. ICC/IF: NIH3T3 and SV40LT-SMC cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab8227 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-Fr Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
WB 1/1000 - 1/5000. Can be blocked with Human beta Actin peptide (ab13772).
ICC Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

IHC-FrFl Use at an assay dependent concentration.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IHC-P Use a concentration of 0.2 µg/ml.
ICC/IF Use a concentration of 1 µg/ml.
ELISA 1/1000.

Target

Anti-beta Actin antibody images

  • This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Milk before being incubated with ab8227 overnight at 4°C. Antibody binding was detected using ab175781 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

  • ab8227 staining beta Actin in SV40LT-SMC cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab8227 at 1μg/ml (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • IHC image of ab8227 staining beta Actin in rat small intestine formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with EDTA (epitope retrieval solution 2) for 20 mins. The section was then incubated with ab8227, 0.2μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.



  • Performed under reducing conditions.

    Exposure time : 1 minute

    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab8227 overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP secondary antibody, and visualised using ECL development solution ab133406

  • ab8227 staining beta Actin in NIH3T3 cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab8227 at 1μg/ml (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • IHC image of beta actin staining in a section of formalin-fixed paraffin-embedded normal human colon*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was then incubated with ab8227, 1/1000 dilution, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody (ab6720, 1/1000 dilution) was used to detect the primary, and visualized using an HRP conjugated ABC system. Streptavidin HRP was used, ab7403 at a 1/10000 dilution. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was then counterstained with haematoxylin and mounted with DPX.

    The inset negative control image is taken from an identical assay without primary antibody.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre



  • Exposure time : 5 seconds

    All lanes : Anti-beta Actin antibody (ab8227) at 1/1000 dilution

    Lane 1 : HeLa nuclear lysate at 20 µg/ml
    Lane 2 : HeLa whole cell lysate at 20 µg/ml
    Lane 3 : A431 cell lysate at 20 µg/ml
    Lane 4 : Jurkat cell lysate at 20 µg/ml
    Lane 5 : HEK 293 cell lysate at 20 µg/ml
    Lane 6 : HeLa nuclear lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 7 : HeLa whole cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 8 : A431 cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 9 : Jurkate cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 10 : HEK 293 cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

  • ICC/IF image of ab8227 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab8227, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue).

  • IHC image of beta Actin staining in normal human colon, formalin-fixed and paraffin-embedded tissue*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab8227, 3µg/ml overnight at +4°C. A anti-rabbit HRP secondary antibody (Ab97200, 1/200 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

    The inset negative control image is taken from an identical assay without primary antibody.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • All lanes : Anti-beta Actin antibody (ab8227) at 1/1000 dilution

    Lane 1 : HeLa nuclear lysate at 20 µg/ml
    Lane 2 : HeLa whole cell lysate at 20 µg/ml
    Lane 3 : A431 cell lysate at 20 µg/ml
    Lane 4 : Jurkat cell lysate at 20 µg/ml
    Lane 5 : HEK 293 cell lysate at 20 µg/ml
    Lane 6 : HeLa nuclear lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 7 : HeLa whole cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 8 : A431 cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 9 : Jurkate cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 10 : HEK 293 cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution



  • Exposure time : 30 seconds

    Western blot using ab8227 at 1/1000.

    Lysates:
    Lane 1: HeLa cells (Human)
    Lane 2: 3T3 cells (Mouse)
    Lane 3: Fish Liver
    Lane 4: Rabbit Liver
    Lane 5: MDCK cells (Dog)
    Lane 6: EBTr cells (Cow)
    Lane 7: SL-29 cells (Chicken)
    Lane 8: CHO cells (Chinese Hamster)
    Lane 9: Xenopus embryo

    Secondary ab: anti-rabbit HRP (ab6721)
    Exposure time: 30 sec
    Lysates at 20µg/lane.
    Expected molecular weight: 41.7kDa.

  • ab8227 staining beta Actin in human stomach tissue sections by Immunohistochemistry (frozen sections). Tissue was fixed with acetone and then blocked with 5% serum for 1 hour at 23°C followed by incubation with the primary antibody, at 1µg/ml, for 1 hour at 23°C. A diluted anti-rabbit HRP-conjugated goat polyclonal was used as secondary antibody.

    See Abreview

  • IHC image of beta Actin staining in human liver FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab8227, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  • ab8227 used in Direct ELISA in NIH 3T3 murine fibroblasts. Primary antibody used at a 1/1000 dilution for 16 hours at 4°C. The secondary antibody used is an AP-conjugated Goat anti-rabbit used at a 1/2000 dilution. A blocking step was performed using 5% BSA for 1 hour at 23°C.

    See Abreview

References for Anti-beta Actin antibody (ab8227)

This product has been referenced in:
  • Lood C  et al. Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease. Nat Med N/A:N/A (2016). WB . Read more (PubMed: 26779811) »
  • Baudot AD  et al. Using enhanced-mitophagy to measure autophagic flux. Methods 75:105-11 (2015). WB . Read more (PubMed: 25498004) »

See all 306 Publications for this product

Product Wall

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (10)
Sample Zebrafish Tissue lysate - whole (Larvae 5hpf)
Specification Larvae 5hpf
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Mr. Pedro Guedes Dias

Verified customer

Submitted Aug 22 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (10)
Sample Rat Cell lysate - whole cell (Primary Neurons)
Specification Primary Neurons
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted Aug 22 2014

Application Immunohistochemistry (Frozen sections)
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Sample Mouse Tissue sections (Skin)
Specification Skin
Permeabilization Yes - 0,3% TritonX
Fixative Perfusion fixed with paraformaldehyde and picric acid
Username

Mr. Frank Rommel

Verified customer

Submitted Aug 08 2014

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing (Biorad Criterion TGX Stain Free Gel, Any kD, Cat. number 5678123)
Sample Fruit fly (Drosophila melanogaster) Tissue lysate - whole (Drosophila heads)
Specification Drosophila heads
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted May 02 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (12%)
Sample Rat Tissue lysate - whole (ovaries)
Specification ovaries
Blocking step Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Username

Mrs. Henda Nabli

Verified customer

Submitted Oct 01 2013

I just received an update form lab regarding the epitope for ab79667 and ab113279. Unfortunately the epitope has not been determined so the information is not available. I am sorry we will not be able to suggest these to be paired with ab8227 in sELISA.

I have had the opportunity to discuss our QC methods regarding this product with the lab. Ab17721 is batch tested by us in WB in HeLa, Jurkat and NIH3T3 lysates and also in Immunofluorescence in HeLa cells. Each new AP purified must pass both of these ...

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Mouse Tissue lysate - whole (Lung)
Gel Running Conditions Reduced Denaturing
Loading amount 14 µg
Specification Lung
Blocking step Odyssey TBS Blocking Buffer (proprietary) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 23°C
Username

Pete Serbedzija

Verified customer

Submitted Apr 29 2016

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Mouse Cell lysate - whole cell (liver, hepatocytes)
Specification liver, hepatocytes
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Feb 09 2015

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 5% · Temperature: 21°C
Sample Zebrafish Cell (Whole Body)
Specification Whole Body
Permeabilization Yes - Triton X-100
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Nov 25 2014

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"